PubMed · 15024161
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Nathan Mise, Philip Avner. 2004. BAC modification using a RecA expressing shuttle vector system.. https://doi.org/10.1385/1-59259-753-x%3A077
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Bacterial artificial chromosome (BAC) is widely used to manipulate herpesvirus genome and generate recombinant virus. Here, we developed a new KSHV BACmid, namely as iBAC, by replacing the EGFP with TET3G transactivator under EF1α promoter and inserted Tet response elements in the promoter of RTA in the original KSHV BAC16 clone and characterized KSHV lytic replication in SLK-iBAC cells. SLK-iBAC cells developed more efficient lytic replication and generated more progeny virus than iSLK-BAC16 cells upon the same conditions of doxycycline treatment. Since SLK-iBAC cells only occupied hygromycin selection marker, it is convenient to generate cellular gene knockout via lentivirus-mediated CRISPR-Cas9 or stably express viral or cellular gene via lentivirus followed by antibiotic selection, making iBAC system a better tool to identify cellular targets of viral proteins in the context of virus infection or study the role of viral or cellular genes for KSHV lytic replication and pathogenesis. In addition, iBAC is color-free and can be utilized to track subcellular localization of viral proteins or colocalization between different viral proteins by introducing fusing fluorescent proteins into the BAC backbone. Therefore, the new KSHV iBAC is a powerful inducible tool to study KSHV lytic replication and pathogenesis in cell model.
Fine mapping and positional cloning will eventually improve with the anchoring of additional markers derived from genomic clones such as BACs. From 2,603 new BAC-end genomic sequences from Gossypium hirsutum Acala 'Maxxa', 1,316 PCR primer pairs (designated as MUSB) were designed to flank microsatellite or simple sequence repeat motif sequences. Most (1164 or 88%) MUSB primer pairs successfully amplified DNA from three species of cotton with an average of three amplicons per marker and 365 markers (21%) were polymorphic between G. hirsutum and G. barbadense. An interspecific RIL population developed from the above two entries was used to map 433 marker loci and 46 linkage groups with a genetic distance of 2,126.3 cM covering approximately 45% of the cotton genome and an average distance between two loci of 4.9 cM. Based on genome-specific chromosomes identified in G. hirsutum tetraploid (A and D), 56.9% of the coverage was located on the A subgenome while 39.7% was assigned to the D subgenome in the genetic map, suggesting that the A subgenome may be more polymorphic and recombinationally active than originally thought. The linkage groups were assigned to 23 of the 26 chromosomes. This is the first genetic map in which the linkage groups A01 and A02/D03 have been assigned to specific chromosomes. In addition the MUSB-derived markers from BAC-end sequences markers allows fine genetic and QTL mapping of important traits and for the first time provides reconciliation of the genetic and physical maps. Limited QTL analyses suggested that loci on chromosomes 2, 3, 12, 15 and 18 may affect variation in fiber quality traits. The original BAC clones containing the newly mapped MUSB that tag the QTLs provide critical DNA regions for the discovery of gene sequences involved in biological processes such as fiber development and pest resistance in cotton.
As part of an initiative to develop Brachypodium distachyon as a genomic "bridge" species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.